Acrosin from human spermatozoa was required for studies of immunological interference with fertilization, but not detailed purification scheme was available for the human enzyme. Since human semen samples cannot be obtained cheaply or in large numbers and contain relatively small amounts of acrosin, development of purification procedures was carried out with bovine semen. Bovine acrosin had not previously been fully purified, and over 1 mg of pure acrosin was obtained from 100 mL of bovine semen, by a process of saline and Triton X-100 washes of the spermatozoa, 1 mM HCl extraction, gel filtration, and ion-exchange and affinity chromatography. The bovine acrosin had a molecular weight (MW) of 39 000 and a specific activity of 93 U/mg, measured with 0.5 mM benzoyl arginine ethyl ester. The same extraction procedure could be followed for human acrosin, but better yields were obtained in the purification if the ion-exchange step was omitted. The human acrosin had a MW of 49 000, and traces of a 38 000 MW component were sometimes observed. From 14 human semen samples, containing initially 7-10 U of acrosin activity, about 2.5 U (approximately 20 micrograms of protein) could be obtained in a pure state.